Methods for reducing cd36 expression

ABSTRACT

The invention provides a method for treating one or more complications of diabetes in a mammal. The method comprises administering to a mammal in need thereof an effective amount of an aromatic-cationic peptide having at least one net positive charge; a minimum of four amino acids; a maximum of about twenty amino acids; a relationship between the minimum number of net positive charges (pm) and the total number of amino acid residues (r) wherein 3 pm is the largest number that is less than or equal to r+1; and a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (pt) wherein 2a is the largest number that is less than or equal to pt+1, except that when a is 1, pt may also be 1.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/140,325, filed Sep. 24, 2018, which is a continuation of U.S.application Ser. No. 15/369,281, filed Dec. 5, 2016, which is acontinuation of U.S. application Ser. No. 14/285,226, filed May 22,2014, which is a continuation of U.S. application Ser. No. 14/075,686,filed Nov. 8, 2013, now U.S. Pat. No. 9,150,614, issued Oct. 6, 2015,which is a continuation of U.S. application Ser. No. 12/850,079, filedAug. 4, 2010, now U.S. Pat. No. 8,603,971, issued Dec. 10, 2013, whichis a continuation of U.S. application Ser. No. 12/434,216, filed May 1,2009, now U.S. Pat. No. 7,811,987, issued Oct. 12, 2010, which is acontinuation of U.S. application Ser. No. 11/532,764, filed on Sep. 18,2006, now U.S. Pat. No. 7,541,340, issued Jun. 2, 2009, which claimspriority to U.S. Provisional Application Ser. No. 60/718,170, filed onSep. 16, 2005, the contents of which are hereby incorporated byreference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

The invention described in this application was funded by the NationalInstitute of Drug Abuse, Grant No. P01 DA08924, the National Instituteof Neurological Diseases and Stroke, Grant No. R21 NS48295, and theNational Heart, Lung and Blood Institute, Grant No. RO1 HL082511. TheUnited States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

CD36 is a transmembrane protein of the class B scavenger receptorfamily. The protein is widely expressed on numerous cells, such asmicrovascular endothelium, macrophages, platelets, adipocytes,epithelial cells (e.g., intestinal epithelial and renal tubular cells,etc.), pancreatic islet cells and cardiac muscle. The receptor mayinteract with multiple extracellular ligands, such as thrombospondin-1,long-chain fatty acids, and oxidized low-density lipoprotein.

Abnormal expression of CD36 has been implicated in a wide variety ofdiseases and conditions. For example, mice lacking CD36 have lessatherosclerotic lesions when fed a Western diet compared to wild-typemice. Further, CD36 knock out mice were reported to be protected againstacute cerebral ischemia.

Therefore, methods for reducing expression of CD36 expression arebeneficial for treating a disease or condition characterized by abnormalexpression of CD36.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method for reducing CD36expression in a cell. The method comprises contacting the cell with aneffective amount of an aromatic-cationic peptide.

In another embodiment, the invention provides a method for reducing CD36expression in a mammal in need thereof. The method comprisesadministering to the mammal an effective amount of an aromatic-cationicpeptide.

In yet another embodiment, the invention provides a method for treatinga disease or condition characterized by increased CD36 expression in amammal in need thereof. The method comprises administering to the mammalan effective amount of an aromatic-cationic peptide.

In a further embodiment, the invention provides a method for treatingureteral obstruction in a mammal in need thereof. The method comprisesadministering to the mammal an effective amount of an aromatic-cationicpeptide.

In yet a further embodiment, the invention provides a method fortreating diabetic nephropathy in a mammal in need thereof. The methodcomprises administering to the mammal an effective amount of anaromatic-cationic peptide.

In another embodiment, the invention provides a method for reducing CD36expression in a removed organ or tissue. The method comprisesadministering to the mammal an effective amount of an aromatic-cationicpeptide.

The aromatic-cationic peptides useful in the methods of the presentinvention have at least one net positive charge; a minimum of four aminoacids; a maximum of about twenty amino acids; a relationship between theminimum number of net positive charges (μm) and the total number ofamino acid residues (r) wherein 3 μm is the largest number that is lessthan or equal to r+1; and a relationship between the minimum number ofaromatic groups (a) and the total number of net positive charges (p_(t))wherein 2a is the largest number that is less than or equal to p_(t)+1,except that when a is 1, p_(t) may also be 1.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. SS-31 reduced oxLDL-induced CD36 mRNA expression, CD36protein expression, and foam cell formation in mouse peritonealmacrophages.

FIGS. 2A-2B. SS-31 treatment reduced infarct volume and hemisphericswelling in wild-type mice subjected to acute cerebral ischemia.

FIG. 3. SS-31 treatment reduced the decrease in reduced glutathione(GSH) in post-ischemic brain in wild-type mice.

FIG. 4. SS-31 had no effect in reducing infarct volume or hemisphericswelling in CD36 knock-out mice subjected to acute cerebral ischemia.

FIG. 5. SS-31 did not reduce GSH depletion in post-ischemic brain fromCD36 knock-out mice.

FIG. 6. SS-31 reduced CD36 mRNA expression in post-ischemic brain inwild-type mice.

FIGS. 7A-7C. SS-31 decreases CD36 expression on renal tubular cellsafter unilateral ureteral obstruction (UUO). Contralateral unobstructedkidney (FIG. 7A); obstructed kidney in animals treated with saline (FIG.73-B); and obstructed kidneys obtained from rats treated with SS-31(FIG. 7C).

FIGS. 8A-8C. SS-31 reduces lipid peroxidation in kidney after UUO.Tubular cells in the obstructed kidney (FIG. 8B), contralateralunobstructed control (FIG. 8A); obstructed kidneys from rats treatedwith SS-31 (FIG. 8C).

FIGS. 9A-9C. SS-31 reduced tubular cell apoptosis in obstructed kidneyafter UUO. Obstructed kidney from saline-treated animals (FIG. 9B);contralateral unobstructed control (FIG. 9A); obstructed kidney fromSS-31 treated animals (FIG. 9C).

FIGS. 10A-10C. SS-31 reduced macrophage infiltration in obstructedkidney induced by UUO. Obstructed kidney (FIG. 10B); contralateralunobstructed control (FIG. 10A); rats treated with SS-31 (FIG. 10C).

FIGS. 11A-11C. SS-31 reduced interstitial fibrosis in obstructed kidneyafter UUO. Obstructed kidney (FIG. 11B); contralateral unobstructedcontrol (FIG. 11A); rats treated with SS-31 (FIG. 11C).

FIGS. 12A-12J. Cold storage of isolated hearts with SS-31 or SS-20prevented upregulation of CD36 expression. The “background” control(FIGS. 12A and 12B) represents two sections from a normal non-ischemicheart that were not treated with the primary anti-CD-36 antibody.“Normal heart” (FIGS. 12C and 12D) represents two sections obtained froma non-ischemic heart. The sections from a representative heart stored inSt. Thomas solution (FIGS. 12E and 12F) for 18 hours at 4° C. showedincreased CD36 staining compared to “Normal heart.” CD36 staining wassignificantly reduced in hearts stored with either 1 nM SS-31 (FIGS. 12Gand 12H) or 100 nM SS-20 (FIGS. 12I and 12J) in St. Thomas solution.

FIGS. 13A-13D. SS-31 and SS-20 reduced lipid peroxidation in isolatedguinea pig hearts subjected to warm reperfusion after prolonged coldischemia. HNE staining in hearts subjected to 18 hours of cold storagein St. Thomas solution (FIG. 13B) compared to non-ischemic hearts (FIG.13A). HNE staining was reduced in hearts stored in SS-31 (FIG. 913C) orSS-20 (FIG. 13D).

FIGS. 14A-14H. SS-31 and SS-20 abolished endothelial apoptosis inisolated guinea pig hearts subjected to warm reperfusion after prolongedcold ischemia. Hearts subjected to 18 hours of cold storage in St.Thomas solution (FIGS. 14C and 14D); non-ischemic normal hearts (FIGS.14A and 14B). Apoptotic cells were not observed in hearts stored inSS-31 (FIGS. 14E and 14F) or SS-20 (FIGS. 14G and 14H).

FIGS. 15A-15B. SS-31 and SS-20 preserves coronary flow in isolatedguinea pig hearts subjected to warm reperfusion after prolonged coldischemia. Guinea pig hearts perfused with a cardioplegic solution (St.Thomas solution) alone or St. Thomas solution containing either 1 nMSS-31 (FIG. 15A) or 100 nM SS-20 (FIG. 15B) for 3 min. and thensubjected to 18 hours of cold ischemia (4° C.).

FIGS. 16A-16C. SS-31 prevented damage to proximal tubules in diabeticmice. Diabetes was induced by streptozotocin (STZ) injection for 5 d.Kidney sections obtained after 3 weeks showed loss of brush border inSTZ-treated animals (FIG. 16A, FIG. 16B) that was not seen in mice nottreated with STZ (FIG. 16A). The loss of brush border was not seen inSTZ-treated animal that received daily SS-31 (3 mg/kg) (FIG. 16C).

FIGS. 17A-17B. SS-31 prevented renal tubular epithelial cell apoptosisin diabetic mice. Diabetes was induced by streptozotocin (STZ) injectionfor 5 d. Kidney sections obtained after 3 weeks showed dramatic increasein apoptotic cells in proximal tubules in STZ-treated animals (FIG. 17A,panel b) that was not seen in mice not treated with STZ (FIG. 17A, panela). The STZ-induced apoptosis was not seen in mice that received dailySS-31 (3 mg/kg) (FIG. 17A, panel c). The percent of apoptotic cellscaused by STZ was significantly reduced by SS-31 treatment (FIG. 17B).

DETAILED DESCRIPTION OF THE INVENTION Peptides

The invention is directed to the reduction of CD36 expression by certainaromatic-cationic peptides. The aromatic-cationic peptides arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes.

The aromatic-cationic peptides useful in the present invention include aminimum of three amino acids, and preferably include a minimum of fouramino acids, covalently joined by peptide bonds.

The maximum number of amino acids present in the aromatic-cationicpeptides of the present invention is about twenty amino acids covalentlyjoined by peptide bonds. Preferably, the maximum number of amino acidsis about twelve, more preferably about nine, and most preferably aboutsix. Optimally, the number of amino acids present in the peptides isfour.

The amino acids of the aromatic-cationic peptides useful in the presentinvention can be any amino acid. As used herein, the term “amino acid”is used to refer to any organic molecule that contains at least oneamino group and at least one carboxyl group. Preferably, at least oneamino group is at the position relative to a carboxyl group.

The amino acids may be naturally occurring. Naturally occurring aminoacids include, for example, the twenty most common levorotatory (L)amino acids normally found in mammalian proteins, i.e., alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (lieu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp) tyrosine (Tyr), and valine (Val).

Other naturally occurring amino acids include, for example, amino acidsthat are synthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acids ornithine and citrulline aresynthesized in mammalian metabolism during the production of urea.Another example of a naturally occurring amino acid includehydroxyproline (Hyp).

The peptides useful in the present invention optionally contain one ormore non-naturally occurring amino acids. Optimally, the peptide has noamino acids that are naturally occurring. The non-naturally occurringamino acids may be levorotary (L-), dextrorotatory (D-), or mixturesthereof.

Non-naturally occurring amino acids are those amino acids that typicallyare not synthesized in normal metabolic processes in living organisms,and do not naturally occur in proteins. In addition, the non-naturallyoccurring amino acids useful in the present invention preferably arealso not recognized by common proteases.

The non-naturally occurring amino acid can be present at any position inthe peptide. For example, the non-naturally occurring amino acid can beat the N-terminus, the C-terminus, or at any position between theN-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 5′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide usefulin the methods of the present invention is the derivatization of acarboxyl group of an aspartic acid or a glutamic acid residue of thepeptide. One example of derivatization is amidation with ammonia or witha primary or secondary amine, e.g. methylamine, ethylamine,dimethylamine or diethylamine. Another example of derivatizationincludes esterification with for example, methyl or ethyl alcohol.

Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides useful in themethods of the invention should have less than five, preferably lessthan four, more preferably less than three, and most preferably, lessthan two contiguous L-amino acids recognized by common proteases,irrespective of whether the amino acids are naturally or non-naturallyoccurring. Optimally, the peptide has only D-amino acids, and no L-aminoacids.

If the peptide contains protease sensitive sequences of amino acids, atleast one of the amino acids is preferably a non-naturally occurringD-amino acid, thereby conferring protease resistance. An example of aprotease sensitive sequence includes two or more contiguous basic aminoacids that are readily cleaved by common proteases, such asendopeptidases and trypsin. Examples of basic amino acids includearginine, lysine and histidine.

It is important that the aromatic-cationic peptides have a minimumnumber of net positive charges at physiological pH in comparison to thetotal number of amino acid residues in the peptide. The minimum numberof net positive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r).

The minimum number of net positive charges discussed below are all atphysiological pH. The term “physiological pH” as used herein refers tothe normal pH in the cells of the tissues and organs of the mammalianbody. For instance, the physiological pH of a human is normallyapproximately 7.4, but normal physiological pH in mammals may be any pHfrom about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH.

In one embodiment of the present invention, the aromatic-cationicpeptides have a relationship between the minimum number of net positivecharges at physiological pH (p_(m)) and the total number of amino acidresidues (r) wherein 3 p_(m) is the largest number that is less than orequal to r+1. In this embodiment, the relationship between the minimumnumber of net positive charges (p_(m)) and the total number of aminoacid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 33 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2 p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

(r) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 55 6 6 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a).

Naturally occurring amino acids that have an aromatic group include theamino acids histidine, tryptophan, tyrosine, and phenylalanine. Forexample, the hexapeptide Lys-Gln-Tyr-Arg-Phe-Trp has a net positivecharge of two (contributed by the lysine and arginine residues) andthree aromatic groups (contributed by tyrosine, phenylalanine andtryptophan residues).

In one embodiment of the present invention, the aromatic-cationicpeptides useful in the methods of the present invention have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges at physiological pH (p_(t)) wherein3a is the largest number that is less than or equal to p_(t)+1, exceptthat when p_(t) is 1, a may also be 1. In this embodiment, therelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) is as follows:

(p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 1 1 22 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

(p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (a) 1 1 2 2 33 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are preferably amidated with, for example, ammonia to formthe C-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group.

The free carboxylate groups of the asparagine, glutamine, aspartic acid,and glutamic acid residues not occurring at the C-terminus of thearomatic-cationic peptides of the present invention may also be amidatedwherever they occur within the peptide. The amidation at these internalpositions may be with ammonia or any of the primary or secondary aminesdescribed above.

In one embodiment, the aromatic-cationic peptide useful in the methodsof the present invention is a tripeptide having two net positive chargesand at least one aromatic amino acid. In a particular embodiment, thearomatic-cationic peptide useful in the methods of the present inventionis a tripeptide having two net positive charges and two aromatic aminoacids.

Aromatic-cationic peptides useful in the methods of the presentinvention include, but are not limited to, the following peptideexamples:

Lys-D-Arg-Tyr-NH₂, Phe-D-Arg-His, D-Tyr-Trp-Lys-NH₂,Trp-D-Lys-Tyr-Arg-NH₂, Tyr-His-D-Gly-Met, Phe-Arg-D-His-Asp,Tyr-D-Arg-Phe-Lys-Glu-NH₂, Met-Tyr-D-Lys-Phe-Arg,D-His-Glu-Lys-Tyr-D-Phe-Arg, Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂,Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His, Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂,Va1-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂,Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂,Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys,Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂,D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg- Trp-NH₂,Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His- Phe,Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His- Phe-NH₂,Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D- Tyr-Thr,Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr- His-Lys,Glu-Arg-D-Lys-Tyr-D-Va1-Phe-D-His-Trp-Arg-D-Gly- Tyr-Arg-D-Met-NH₂,Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys- D-Phe-Tyr-D-Arg-Gly,D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂,Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe,His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂,Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp,  andThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg- Tyr-Lys-NH₂.

In one embodiment, the peptides useful in the methods of the presentinvention have mu-opioid receptor agonist activity (i.e., they activatethe mu-opioid receptor). Activation of the mu-opioid receptor typicallyelicits an analgesic effect.

In certain instances, an aromatic-cationic peptide having mu-opioidreceptor agonist activity is preferred. For example, during short-termtreatment, such as in an acute disease or condition, it may bebeneficial to use an aromatic-cationic peptide that activates themu-opioid receptor. Such acute diseases and conditions are oftenassociated with moderate or severe pain. In these instances, theanalgesic effect of the aromatic-cationic peptide may be beneficial inthe treatment regimen of the human patient or other mammal. Anaromatic-cationic peptide which does not activate the mu-opioidreceptor, however, may also be used with or without an analgesic,according to clinical requirements.

Alternatively, in other instances, an aromatic-cationic peptide thatdoes not have mu-opioid receptor agonist activity is preferred. Forexample, during long-term treatment, such as in a chronic disease stateor condition, the use of an aromatic-cationic peptide that activates themu-opioid receptor may be contraindicated. In these instances thepotentially adverse or addictive effects of the aromatic-cationicpeptide may preclude the use of an aromatic-cationic peptide thatactivates the mu-opioid receptor in the treatment regimen of a humanpatient or other mammal. Potential adverse effects may include sedation,constipation and respiratory depression. In such instances anaromatic-cationic peptide that does not activate the mu-opioid receptormay be an appropriate treatment.

Peptides useful in the methods of the present invention which havemu-opioid receptor agonist activity are typically those peptides whichhave a tyrosine residue or a tyrosine derivative at the N-terminus(i.e., the first amino acid position). Preferred derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′, 6′-dimethyltyrosine (2′6′Dmt); 3′,5′-dimethyltyrosine (3′5′Dmt); N,2′, 6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In a particular preferred embodiment, a peptide that has mu-opioidreceptor agonist activity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (forconvenience represented by the acronym: DALDA, which is referred toherein as SS-01). DALDA has a net positive charge of three, contributedby the amino acids tyrosine, arginine, and lysine and has two aromaticgroups contributed by the amino acids phenylalanine and tyrosine. Thetyrosine of DALDA can be a modified derivative of tyrosine such as in2′, 6′-dimethyltyrosine to produce the compound having the formula 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (i.e., Dmt′-DALDA), which is referred to hereinas SS-02).

Peptides that do not have mu-opioid receptor agonist activity generallydo not have a tyrosine residue or a derivative of tyrosine at theN-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine.

In one embodiment, the amino acid at the N-terminus is phenylalanine orits derivative. Preferred derivatives of phenylalanine include2-methylphenylalanine (Mmp), 2′, 6′-dimethylphenylalanine (Dmp), N,2′,6′-trimethylphenylalanine (Tmp), and 2′-hydroxy-6′-methylphenylalanine(Hmp).

Another aromatic-cationic peptide that does not have mu-opioid receptoragonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂ (i.e.,Phe′-DALDA, which is referred to herein as SS-20). Alternatively, theN-terminal phenylalanine can be a derivative of phenylalanine such as2′, 6′-dimethylphenylalanine (2′6′Dmp). DALDA containing 2′,6′-dimethylphenylalanine at amino acid position 1 has the formula 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂, (i.e. 2′6′Dmp′-DALDA).

In a preferred embodiment, the amino acid sequence of Dmt′-DALDA (SS-02)is rearranged such that Dmt is not at the N-terminus. An example of suchan aromatic-cationic peptide that does not have mu-opioid receptoragonist activity has the formula D-Arg-2′6′Dmt-Lys-Phe-NH₂ (referred toin this specification as SS-31).

DALDA, Phe′-DALDA, SS-31, and their derivatives can further includefunctional analogs. A peptide is considered a functional analog ofDALDA, Phe′-DALDA, or SS-31 if the analog has the same function asDALDA, Phe′-DALDA, or SS-31. The analog may, for example, be asubstitution variant of DALDA, Phe′-DALDA, or SS-31, wherein one or moreamino acids are substituted by another amino acid.

Suitable substitution variants of DALDA, Phe′-DALDA, or SS-31 includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physiochemical characteristics as follows:

-   -   (a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);    -   (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);    -   (c) Basic amino acids: His(H4) Arg(R) Lys(K);    -   (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and    -   (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physiological characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of analogs useful in the practice of the present invention thatactivate mu-opioid receptors include, but are not limited, to thearomatic-cationic peptides shown in Table 1.

TABLE 1 Amino Amino Amino Amino Amino Acid C-Ter- Acid Acid Acid AcidPosition minal Position Position Position Position 5 (if Modifi- 1 2 3 4present) cation Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-ArgPhe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′DmtD-Arg Phe Lys Cys NH₂ 2′6′Dmt D-Arg Phe Lys- NH₂ NH(CH₂)₂—NH- dns2′6′Dmt D-Arg Phe Lys- NH₂ NH(CH₂)₂—NH- atn 2′6′Dmt D-Arg Phe dnsLys NH₂2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg PheOrn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′DmtD-Arg Phe Ahp (2- NH₂ aminoheptanoic acid) Bio- D-Arg Phe Lys NH₂2′6′Dmt 3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′DmtD-Arg Phe Dab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ TyrD-Arg Tyr Orn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′DmtD-Arg Tyr Lys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂2′6′Dmt D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg2′6′Dmt Orn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt DapNH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′DmtD-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe DapNH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂2′6′Dmt D-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys PheArg NH₂ 2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′DmtD-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ TyrD-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt DabNH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′DmtD-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′DmtOrn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-DapPhe Arg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of analogs useful in the practice of the present invention thatdo not activate mu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 2.

TABLE 2 Amino Acid Amino Acid Amino Acid Amino Acid C-Terminal Position1 Position 2 Position 3 Position 4 Modification D-Arg Dmt Lys Phe NH₂D-Arg Dmt Phe Lys NH₂ D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-ArgLys Dmt Phe NH₂ D-Arg Lys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe LysD-Arg Dmt NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha = cyclohexyl

The amino acids of the peptides shown in table 1 and 2 may be in eitherthe L- or the D-configuration.

Methods

The aromatic-cationic peptides described above are useful in reducingCD36 expression in a cell. For the purposes of this specification CD36expression in a cell is considered to be reduced if the expression ofCD36 is decreased by about 10%, preferably by about 25%, more preferablyby about 50%, even more preferably by about 75%. Optimally, CD36 isreduced to about normal levels in a cell.

CD36 is expressed on a wide variety of cells. Examples of such cellsinclude macrophages, platelets, adipocytes, endothelial cells such asmicrovascular endothelial cells and umbilical vein endothelial cells;epithelial cells such as intestinal epithelial cells, gall bladderepithelial cells, bladder epithelial cells, bronchial epithelial cellsand alvelolar epithelial cells; renal tubular cells; pancreatic isletcells; hepatocytes; skeletal muscle cells; cardiac muscle cells;neuronal cells; glia cells; pancreas cells; sperm cells; etc.

For the purposes of this specification, cells expressing about 10%,typically about 25%, about typically about 50%, and even more typicallyabout 75% more CD36 than normal cells are considered to expressincreased levels of CD36.

In one embodiment, the invention provides a method for reducing CD36expression in a cell. Any cell that expresses CD36 can be used in themethod of the invention, and include those mentioned above. The methodfor reducing CD36 expression in a cell comprises contacting the cellwith an effective amount of an aromatic-cationic peptide describedabove.

In another embodiment, the invention provides a method for reducing CD36expression in a mammal in need thereof. The method for reducing CD36expression in the mammal comprises administering to the mammal aneffective amount of an aromatic-cationic peptide described herein.

Mammals in need of reducing CD36 expression include, for example,mammals that have increased CD36 expression. The increased expression ofCD36 is associated with various diseases and conditions. Examples ofdiseases and conditions characterized by increased CD36 expressioninclude, but are not limited to, atherosclerosis, inflammation, abnormalangiogenesis, abnormal lipid metabolism, abnormal removal of apoptoticcells, ischemia such as cerebral ischemia and myocardial ischemia,ischemia reperfusion, ureteral obstruction, stroke, Alzheimer's Disease,diabetes, diabetic nephropathy and obesity. A discussion on theinvolvement of CD36 in atherosclerosis may be found in “Targeteddisruption of the class B scavenger receptor CD36 protects againstatherosclerotic lesion development in mice,” Febbraio M, Podrez E A,Smith J D, Hajjar D P, Hazen S L et al., J Clinical Investigation,105:1049-1056, 2000, and “CD36: a class B scavenger receptor involved inangiogenesis, atherosclerosis, inflammation, and lipid metabolism,”Febbraio M., Hajjar D P and Silverstein R L, Journal of ClinicalInvestigation, 108:785-791, 2001.

Mammals in need of reducing CD36 expression also include mammalssuffering from complications of diabetes. Some complications of diabetesinclude, in addition to nephropathy, neuropathy, retinopathy, coronaryartery disease, and peripheral vascular disease associated withdiabetes.

In another embodiment, the invention relates to a method for reducingCD36 expression in removed organs and tissues. The method comprisescontacting the removed organ or tissue with an effective amount of anaromatic-cationic peptide described above. An organ or tissue may, forexample, be removed from a donor for autologous or heterologoustransplantation. Some examples of organs and tissues include heart,lungs, pancreas, kidney, liver, skin, etc.

Synthesis of the Peptides

The peptides useful in the methods of the present invention may besynthesized by any of the methods well known in the art. Suitablemethods for chemically synthesizing the protein include, for examplethose described by Stuart and Young in “Solid Phase Peptide Synthesis,”Second Edition, Pierce Chemical Company (1984), and in “Solid PhasePeptide Synthesis,” Methods Enzymol., 289, Academic Press, Inc, New York(1997).

Modes of Administration

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods.

In vitro methods typically include cultured samples. For example, a cellcan be placed in a reservoir (e.g., tissue culture plate), and incubatedwith an aromatic-cationic peptide under appropriate conditions suitablefor reducing CD36 expression. Suitable incubation conditions can bereadily determined by those skilled in the art.

Ex vivo methods typically include cells, organs or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the peptide under appropriate conditions. Thecontacted cells, organs or tissues are normally returned to the donor,placed in a recipient, or stored for future use. Thus, the peptide isgenerally in a pharmaceutically acceptable carrier.

In vivo methods are typically limited to the administration of anaromatic-cationic peptide, such as those described above, to a mammal,preferably a human. The peptides useful in the methods of the presentinvention are administered to a mammal in an amount effective inreducing expression CD36 or treating the mammal. The effective amount isdetermined during pre-clinical trials and clinical trials by methodsfamiliar to physicians and clinicians.

An effective amount of a peptide useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Thepeptide may be administered systemically or locally.

In one embodiment, the peptide is administered intravenously. Forexample, the aromatic-cationic peptides useful in the methods of thepresent invention may be administered via rapid intravenous bolusinjection. Preferably, however, the peptide is administered as aconstant rate intravenous infusion.

The peptide may also be administered orally, topically, intranasally,intramuscularly, subcutaneously, or transdermally. In a preferredembodiment, transdermal administration of the aromatic-cationic peptidesby methods of the present invention is by iontophoresis, in which thecharged peptide is delivered across the skin by an electric current.

Other routes of administration include intracerebroventricularly orintrathecally. Intracerebroventiculatly refers to administration intothe ventricular system of the brain. Intrathecally refers toadministration into the space under the arachnoid membrane of the spinalcord. Thus intracerebroventricular or intrathecal administration may bepreferred for those diseases and conditions which affect the organs ortissues of the central nervous system.

The peptides useful in the methods of the invention may also beadministered to mammals by sustained release, as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum or plasma concentration.

A description of methods for delivering a compound by controlled releasecan be found in international PCT Application No. WO 02/083106. The PCTapplication is incorporated herein by reference in its entirety.

Any formulation known in the art of pharmacy is suitable foradministration of the aromatic-cationic peptides useful in the methodsof the present invention. For oral administration, liquid or solidformulations may be used. Some examples of formulations include tablets,gelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like. The peptides can be mixed with a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

For systemic, intracerebroventricular, intrathecal, topical, intranasal,subcutaneous, or transdermal administration, formulations of thearomatic-cationic peptides useful in the methods of the presentinventions may utilize conventional diluents, carriers, or excipientsetc., such as those known in the art to deliver the peptides. Forexample, the formulations may comprise one or more of the following: astabilizer, a surfactant, preferably a nonionic surfactant, andoptionally a salt and/or a buffering agent. The peptide may be deliveredin the form of an aqueous solution, or in a lyophilized form.

The stabilizer may, for example, be an amino acid, such as for instance,glycine; or an oligosaccharide, such as for example, sucrose, tetralose,lactose or a dextran. Alternatively, the stabilizer may be a sugaralcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% weight for weight of the peptide.

The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween20,Tween80; a polyethylene glycol or a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

The salt or buffering agent may be any salt or buffering agent, such asfor example, sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buffering agent maintains the pH of thepharmaceutical composition in the range of about 5.5 to about 7.5. Thesalt and/or buffering agent is also useful to maintain the osmolality ata level suitable for administration to a human or an animal. Preferablythe salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

The formulations of the peptides useful in the methods of the presentinvention may additionally contain one or more conventional additive.Some examples of such additives include a solubilizer such as, forexample, glycerol; an antioxidant such as for example, benzalkoniumchloride (a mixture of quaternary ammonium compounds, known as “quats”),benzyl alcohol, chloretone or chlorobutanol; anaesthetic agent such asfor example a morphine derivative; or an isotonic agent etc., such asdescribed above. As a further precaution against oxidation or otherspoilage, the pharmaceutical compositions may be stored under nitrogengas in vials sealed with impermeable stoppers.

The mammal treated in accordance with the invention can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

EXAMPLES Example 1: SS-31 Reduced Oxidized Low-Density Lipoprotein(oxLDL)-Induced CD36 Expression and Foam Cell Formation in MousePeritoneal Macrophages

Atherosclerosis is thought to develop as a result of lipid uptake byvascular-wall macrophages leading to the development of foam cells andthe elaboration of cytokines and chemokines resulting in smoothmuscle-cell proliferation. CD36 is a scavenger receptor that mediatesuptake of oxLDL into macrophages and subsequent foam-cell development.CD36 knock out mice showed reduced uptake of oxLDL and reducedatherosclerosis.

CD36 expression is regulated at the transcriptional level by variouscellular stimuli, including glucose and oxLDL. Macrophages wereharvested from mice peritoneal cavity and culture overnight in theabsence or presence of oxLDL (50 μg/ml) for 48 h. Incubation with oxLDLsignificantly increased CD36 mRNA (FIG. 1A). Inclusion of SS-31 (10 nMor 1 μM) to the culture medium abolished the up-regulation of CD36 (FIG.1A). SS-31 by itself had no effect on CD36 expression.

Expression of CD36 protein, as determined by western blot, was alsosignificantly increased after 48 h incubation with 25 μg/ml of oxLDL(oxL) when compared to vehicle control (V) (FIG. 1B). Other controlsincluded CD36 expression from mouse heart (H) and macrophages obtainedfrom CD36 knockout mice (KO). The amount of CD36 protein was normalizedto β-actin. Incubation with SS-31 (1 (S) significantly reduced CD36protein expression compared to macrophages exposed to vehicle control(V) (P<0.01, ANOVA with posthoc Neuman Keuls test). Concurrentincubation with SS-31 (1 μM) also significantly inhibited theupregulation of CD36 protein expression in macrophages exposed to 25μg/ml oxLDL for 48 h (oxL/S) (P<0.01, ANOVA with posthoc Neuman Keulstest).

Incubation of macrophages with oxLDL for 48 h also increased foam cellformation (FIG. 1C). Foam cell is indicated by oil red 0 which stainslipid droplets red. Inclusion of SS-31 (1 μM) prevented oxLDL-inducedfoam cell formation (FIG. 1C).

Incubation of macrophages with oxLDL increased apoptotic cells from 6.7%to 32.8%. Concurrent treatment with SS-31 (1 nM) significantly reducedthe percentage of apoptotic cells induced by oxLDL to 20.8%.

Example 2: SS-31 Protected Mice from Acute Cerebral Ischemia

Cerebral ischemia initiates a cascade of cellular and molecular eventsthat lead to brain damage. One such event is postischemic inflammation.Using a mouse model of cerebral ischemia-reperfusion (20 min. occlusionof the middle cerebral artery), it was found that CD36 was upregulatedin microglia and macrophages in the post-ischemic brain, and there wasincreased reactive oxygen species production. CD36 knock out mice had aprofound reduction in reactive oxygen species after ischemia andimproved neurological function compared to wild type mice.

Cerebral ischemia was induced by occlusion of the right middle cerebralartery for 30 min. Wild-type (WT) mice were given either saline vehicle(Veh) (ip, n=19) or SS-31 (2 mg/kg or 5 mg/kg, ip, n=6) at 0, 6, 24 and48 h after ischemia. Mice were killed 3 days after ischemia. Brains wereremoved, frozen, and sectioned. Brain sections were stained by the Nisslstain. Infarct volume and hemispheric swelling was determined using animage analyzer. Data were analyzed by one-way ANOVA with posthocanalysis.

Treatment of wild type mice with SS-31 (2 mg/kg or 5 mg/kg, ip, n=6) at0, 6, 24 and 48 hours after 30 min. occlusion of the middle cerebralartery resulted in a significant reduction in infarct volume (FIG. 2A)and hemispheric swelling (FIG. 2B) compared to saline controls. (*P<0.05compared to Veh).

Thirty min. cerebral ischemia in WT mice resulted in significantdepletion in reduced glutathione (GSH) in the ipsilateral cortex andstriatum compared to the contralateral side in vehicle-treated animals(FIG. 3). The depletion of GSH in the ipsilateral cortex wassignificantly reduced in mice treated with SS-31 (2 mg/kg ip at 0, 6, 24and 48 h) (FIG. 3). The depletion of GSH in the striatum was alsoreduced by SS-31 treatment but did not reach statistical significance.

Example 3: SS-31 Mediated Protection Against Acute Cerebral IschemiaMimics Protection Observed in CD36 Knockout Mice

CD36 knockout (CD36 KO) mice were subjected to acute cerebral ischemiaas described under Example 2. CD36 KO mice were given either salinevehicle (Veh) (ip, n=5) or SS-31 (2 mg/kg, i.p. n=5) at 0, 6, 24 and 48h after 30 min ischemia. Infarct volume (FIG. 4A) and hemisphericswelling (FIG. 4B) in CD36 KO mice were similar whether they receivedsaline or SS-31.

Treatment of CD36 KO mice with SS-31 (2 mg/kg, i.p., n=5) also failed tofurther prevent GSH depletion in the ipsilateral cortex caused by 30 minischemia (FIG. 5).

These data suggest that the protective action of SS-31 against acutecerebral ischemia may be mediated by inhibiting the upregulation ofCD36.

Example 4: SS-31 Reduced CD36 mRNA Expression in Post-Ischemic Brain

Transient occlusion of the middle cerebral artery has been shown tosignificantly increase the expression of CD36 mRNA in microglia andmacrophages in the post-ischemic brain. Wild-type mice were given salinevehicle (Veh, i.p., n=6) or SS-31 (5 mg/kg, i.p., n=6) at 0 and 6 hafter 30 min ischemia, and CD36 mRNA levels were determined using realtime PCR. CD36 expression was upregulated almost 6-fold in theipsilateral brain compared to the contralateral brain in mice thatreceived saline (FIG. 6). CD36 mRNA was significantly reduced in theipsilateral brain in mice that received SS-31 treatment (FIG. 6).

Example 5: SS-31 Suppressed Upregulation of CD-36 in Renal Tubular CellsFollowing Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common clinical disorderassociated with tubular cell apoptosis, macrophage infiltration, andinterstitial fibrosis. Interstitial fibrosis leads to a hypoxicenvironment and contributes to progressive decline in renal functiondespite surgical correction. CD36 has been shown to be expressed onrenal tubular cells.

CD36 was found to have been upregulated in tubular cells after UUO. UUOwas performed in Sprague-Dawley rats. The rats were treated with saline(ip, n=6) or SS-31 (1 mg/kg ip, n=6) one day prior to induction of UUO,and once a day for 14 days after UUO. Rats were killed, kidneys removed,embedded in paraffin and sectioned. The slides were treated with theanti-CD36 polyclonal IgG (Santa Cruz #sc-9154; 1:100 with blockingserum) at room temperature for 1.5 hours. The slides were then incubatedwith the second antibody conjugated with biotin (anti-rabbit IgG-G1; ABCkit, PK-6101) at room temperature for 30 min. The slides were thentreated with avidin, developed with DAB and counterstained with 10%hematoxylin. The contralateral unobstructed kidney served as the controlfor each animal.

UUO resulted in tubular dilation and significant increase in expressionof CD36 on the tubular cells (FIG. 7). Tubular dilation was alsoobserved in rats treated with SS-31, but there was a significantreduction in CD36 expression (FIG. 3). CD36 expression (brown stain) isprimarily found on tubular cells in the contralateral unobstructedkidney (FIG. 7A). CD36 expression was increased in the obstructed kidneyin animals treated with saline (FIG. 7B), but was much reduced inobstructed kidneys obtained from rats treated with SS-31 (FIG. 7C).

To determine whether SS-31 reduces lipid peroxidation in kidney afterUUO, rats were treated with either saline (n=6) or SS-31 (1 mg/kg ip,n=6) one day prior to induction of UUO, and once a day for 14 days UUO.Rats were then killed, kidneys removed, embedded in paraffin andsectioned. Slides were incubated with anti-HNE rabbit IgG and abiotin-linked anti-rabbit IgG was used as secondary antibody. The slideswere developed with DAB. Lipid peroxidation, which was increased by UUO,was reduced by SS-31 treatment (FIG. 8). HNE stain (brown) wassignificantly increased in tubular cells in the obstructed kidney (FIG.8B) compared to the contralateral control (FIG. 8A). Obstructed kidneysfrom rats treated with SS-31 showed significantly less HNE stain (FIG.8C) compared to saline-treated rats (FIG. 8B).

To determine whether SS-31 reduced tubular cell apoptosis in obstructedkidney after UUO, rats were treated with either saline (n=6) or SS-31 (1mg/kg ip, n=6) one day prior to induction of UUO, and once a day for 14days after UUO. Rats were then killed, kidneys removed, embedded inparaffin and sectioned. To quantitate nuclei with fragmented DNA, theTUNEL assay were performed with in situ TUNEL kit (Intergen, Purchase,N.Y.). Slides were developed with DAB and counterstained with 10%hematoxylin. The upregulation of CD36 in saline-treated controlsassociated with tubular cell apoptosis was significantly inhibited bySS-31 treatment (FIG. 9). Compared to the contralateral unobstructedcontrol (FIG. 9A), a significant increase in apoptotic cells wasobserved in the obstructed kidney from saline-treated animals (FIG. 9B).The number of apoptotic cells was significantly reduced in obstructedkidney from SS-31 treated animals (FIG. 9C) (P<0.001; n=6).

Macrophage infiltration (FIG. 10) and interstitial fibrosis (FIG. 11)were also prevented by SS-31 treatment. Rats were treated with eithersaline (n=6) or SS-31 (1 mg/kg ip, n=6) one day prior to induction ofUUO, and once a day for 14 days after UUO. Rats were then killed,kidneys removed, embedded in paraffin and sectioned. Slides were treatedwith monoclonal antibody for ED1 macrophage (1:75; Serotec).Horseradish-peroxidase-linked rabbit anti-mouse secondary antibody(Dako) was used for macrophage detection. Sections were thencounterstained with 10% hematoxylin. The number of macrophages in theobstructed kidney in saline-treated rats (FIG. 10B) was significantlyincreased compared to the contralateral unobstructed control (FIG. 10A).Macrophage infiltration was significantly reduced in rats treated withSS-31 (FIG. 10C) (P<0.05; t-test).

Rats were treated with either saline (n=6) or SS-31 (1 mg/kg ip, n=6)one day prior to induction of UUO, and once a day for 14 days after UUO.Rats were then killed, kidneys removed, embedded in paraffin andsectioned. Slides were stained with hematoxylin and eosin and Masson'strichome for interstitial fibrosis (blue stain). Obstructed kidneys fromsaline-treated rats showed increase fibrosis (FIG. 11B) compared to thecontralateral unobstructed control (FIG. 11A); while obstructed kidneysfrom SS-31 treated rats showed significantly less fibrosis (P<0.05;t-test).

These results show that SS-31 suppresses the upregulation of CD36 onrenal tubular cells induced by UUO.

Example 6: SS-31 and SS-20 Reduced CD36 Expression in Isolated HeartsUpon Reperfusion after Prolonged Cold Ischemic Storage

Organ transplantation requires hypothermic storage of the isolated organfor transport to the recipient. Currently, cardiac transplantation islimited by the short time of cold ischemic storage that can be toleratedbefore coronary blood flow is severely compromised (<4 hours). Theexpression of CD36 in coronary endothelium and cardiac muscles isup-regulated in isolated hearts subjected to prolonged cold ischemicstorage and warm reperfusion.

Isolated guinea pig hearts were perfused with St. Thomas solution alone,or St. Thomas solution containing 1 nM SS-31 or 100 nM SS-20, for 3 min.and then stored in the same solution at 4° C. for 18 hours. Afterischemic storage, hearts were reperfused with 34° C. Kreb-Henseleitsolution for 90 min. Hearts freshly isolated from guinea pigs were usedas controls.

The hearts were fixed in paraffin and sliced for immunostaining with ananti-CD36 rabbit polyclonal antibody. The results are shown in FIG. 12.Two sections are shown for each treatment group. Antibody stainingshowed that CD36 is expressed in endothelium and cardiac muscles innormal hearts. The “background” (FIGS. 12A and 12B) represents twosections from a normal non-ischemic heart that was not treated with theprimary antibody. “Normal heart” (FIGS. 12C and 12D) represents twosections obtained from a non-ischemic heart. The sections from arepresentative heart stored in St. Thomas solution (FIGS. 12E and 12F)for 18 hours at 4° C. showed increased CD36 staining compared to “Normalheart.” CD36 staining was significantly reduced in hearts stored witheither 1 nM SS-31 (FIGS. 12G and 12H) or 100 nM SS-20 (FIGS. 12I and12J) in St. Thomas solution for 18 h.

CD36 staining is increased in hearts that have undergone 18 hours ofcold ischemic storage and warm reperfusion. However, hearts that werestored with either 1 nM SS-31 or 100 nM SS-20 did not show theupregulation of CD36 expression.

Lipid peroxidation in the hearts was also decreased by thearomatic-cationic peptides. Guinea pig hearts were perfused with acardioplegic solution (St. Thomas solution) alone or St. Thomas solutioncontaining either 1 nM SS-31 or 100 nM SS-20 for 3 min. and thensubjected to 18 hours of cold ischemia (4° C.). The hearts were thenreperfused with Krebs Henseleit buffer at 34° C. for 90 min.Immunohistochemical analysis of 4-hydroxynonenol (HNE)-modified proteinsin paraffin sections from tissue slices were performed by incubationwith an anti-HNE antibody (Santa Cruz) and a fluorescent secondaryantibody. HNE staining was significantly increased in hearts subjectedto 18 hours of cold storage in St. Thomas solution (FIG. 13B) comparedto non-ischemic hearts (FIG. 13A). HNE staining was reduced in heartsstored in SS-31 (FIG. 13C) or SS-20 (FIG. 13D).

Further, the peptides dramatically reduced endothelial apoptosis (FIG.14). Guinea pig heats were perfused with a cardioplegic solution (St.Thomas solution) alone or St. Thomas solution containing either 1 nMSS-31 or 100 nM SS-20 for 3 min. and then subjected to 18 hours of coldischemia (4° C.). The hearts were then reperfused with Krebs Henseleitbuffer at 34° C. for 90 min. After deparaffinization, sections wereincubated with deoxynucleotidyl transferase (Tdt) with digoxigenin-dNTPfor 1 hour. The reaction was stopped with terminating buffer. Afluorescent anti-digoxigenin antibody was then applied. Hearts subjectedto 18 hours of cold storage in St. Thomas solution (FIGS. 14C and 14D)showed prominent endothelial apoptosis whereas no endothelial apoptosiswas observed in non-ischemic normal hearts (FIGS. 14A and 14B).Apoptotic cells were not observed in hearts stored in SS-31 (FIGS. 14Eand 14F) or SS-20 (FIGS. 14G and 14H).

A significant improvement of coronary blood flow after prolonged coldischemic storage and warm reperfusion occurred (FIG. 15). Guinea pigshearts were perfused with a cardioplegic solution (St. Thomas solution)alone or St. Thomas solution containing either 1 nM SS-31 (FIG. 15A) or100 nM SS-20) (FIG. 15B) for 3 min. and then subjected to 18 hours ofcold ischemia (4° C.). The hearts were then reperfused with KrebsHenseleit buffer at 34° C. for 90 min. Coronary flow was significantlyreduced after prolonged ischemia compared to pre-ischemic control(expressed as 100%). Preservation in either SS-31 or SS-20 significantlyrestored coronary flow to approximately 80% of pre-ischemic flow.

Example 7: SS-31 Prevented Renal Damage in Diabetic Mice

CD36 expression is upregulated in a variety of tissues of diabeticpatients, including monocytes, heart, kidneys, and plasma. High glucoseis known to upregulate the expression of CD36 by improving thetranslational efficiency of CD36 mRNA. Diabetic nephropathy is a commoncomplication of type 1 and type 2 diabetes, and is associated withtubular epithelial degeneration and interstitial fibrosis. CD36 has beenidentified as a mediator of tubular epithelial apoptosis in diabeticnephropathy. High glucose stimulates CD36 expression and apoptosis inproximal tubular epithelial cells.

Streptozotocin (STZ) was used to induce diabetes in mice. Three groupsof CD-1 mice were studied. Group I—no STZ treatment; Group II—STZ (50mg/kg, ip) was given once a day for 5 d; Group III—STZ (50 mg/kg, ip)was given once a day for 5 d, and SS-31 (3 mg/kg, ip) was given once aday for 16 d. STZ treatment resulted in progressive increase in bloodglucose. By week 3, blood glucose values were: Group I (10.6.noteq.0.27mmol/L); Group II (24.5.noteq.1.15 mmol/L); Group III (21.3 1.48mmol/L). Animals were sacrificed after 3 weeks and kidney tissuespreserved for histopathology. Kidney sections were examined by PeriodicSchiff (PAS) staining for renal tubular brush border.

STZ treatment caused dramatic loss of brush border in proximal tubulesin the renal cortex (FIG. 16). In mice not treated with STZ, the renalbrush border in the cortex was stained red with PAS (FIG. 16A, see whitearrows). In mice treated with STZ, the brush border was obliterated, andthe tubular epithelial cells showed small condensed nuclei (FIG. 16B).Daily treatment with SS-31 (3 mg/kg, ip) presented the loss of brushborder in the STZ-treated mice (FIG. 16C), and the nuclei appearednormal (FIG. 16, top and bottom panels). In general, the architecture ofthe proximal renal tubules was preserved in diabetic mice treated withSS-31.

STZ treatment induced significant apoptosis in tubular epithelial cells(FIG. 17). Kidney sections were examined for apoptosis using the TUNELassay. After deparaffinization, sections were incubated withdeoxynucleotidyl transferase (Tdt) with digoxigenin-dNTP for 1 hour. Thereaction was stopped with terminating buffer. A fluorescentanti-digoxigenin antibody was then applied. Kidney sections from micetreated with STZ showed large number of apoptotic nuclei in the proximaltubules (PT) (FIG. 17A, panel b), compared to no apoptotic cells in micenot treated STZ (FIG. 17A, panel a). Treatment with daily SS-31dramatically reduced apoptotic cells in the proximal tubule (FIG. 17A,panel c). FIG. 17B shows the significant decrease in tubular cellapoptosis provided by SS-31.

CD36 expression in proximal tubular epithelial cells is known to beincreased by high glucose and is upregulated in diabetic models. SS-31.by reducing CD36 expression, was able to inhibit tubular cell apoptosisand loss of brush border in mice treated with STZ without affectingblood glucose.

What is claimed is:
 1. A method for treating one or more complicationsof diabetes in a mammal in need thereof, the method comprisingadministering to the mammal an effective amount of an aromatic-cationicpeptide having: (a) at least one net positive charge; (b) a minimum offour amino acids; (c) a maximum of about twenty amino acids; (d) arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 3 p_(m) is thelargest number that is less than or equal to r+1; and (e) a relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges (p_(t)) wherein 2a is the largest number that isless than or equal to _(pt)+1, except that when a is 1, p_(t) may alsobe
 1. 2. The method of claim 1, wherein the one or more complications ofdiabetes is diabetic nephropathy.
 3. The method of claim 1, wherein theone or more complications of diabetes is diabetic neuropathy.
 4. Themethod of claim 1, wherein the one or more complications of diabetes isincreased body weight in the mammal compared to a mammal not sufferingfrom diabetes.
 5. The method of claim 1, wherein the one or morecomplications of diabetes is increased blood glucose compared to amammal not suffering from diabetes.
 6. The method of claim 1, whereinthe peptide has the formula D-Arg-2′6′-Dmt-Lys-Phe-NH₂.
 7. The method ofclaim 1, wherein the peptide has the formula Phe-D-Arg-Phe-Lys-NH₂. 8.The method of claim 1, wherein the mammal is a human.
 9. The method ofclaim 1, wherein the mammal is suffering from type II diabetes.
 10. Themethod of claim 1, wherein the peptide is administered orally,topically, systemically, intravenously, subcutaneously, orintramuscularly.